Throughout this application, various references are referenced by arabic numbers within parenthesis. Full citations for these references may be found at the end of the specification, immediately preceding the claims. These references, in their entireties, are hereby incorporated by reference to more fully describe the state of the art to which this invention pertains.
The present invention is directed to the treatment of central nervous system (CNS) diseases by antibodies.
The nervous system of vertebrates is divided into the central nervous system, comprised of the brain and spinal cord, and the peripheral nervous system, consisting of the outlying nerves (16). The axons of most nerve cells are covered with a myelin sheath, a stack of specialized plasma membranes. Glial cells that wrap around the axons produce the myelin sheath. In the CNS, these cells are called oligodendrocytes. The myelin membranes of the CNS contain myelin basic protein (MBP) and a proteolipid (PLP) that is not found elsewhere in vertebrates. Each region of myelin formed by an individual glial cell is separated from the next region by an unmyelinated area called the node of Ranvier; only at nodes is the axonal membrane in direct contact with the extracellular fluid.
The myelin sheath, which can be 10-12 myelin wraps thick, acts as an electric insulator of the axon by preventing the transfer of ions between the axonal cytoplasm and the extracellular fluids (16). Thus all electric activity in axons is confined to the nodes of Ranvier, the sites where ions can flow across the axonal membrane. Node regions contain a high density of voltage-dependent Na+ channels, about 10,000 per xcexcm2, whereas the regions of axonal membrane between the nodes have few if any channels.
The excess cytosolic positive ions generated at a node during the membrane depolarization associated with an action potential diffuse through the axonal cytoplasm to the next node with very little loss or attenuation because ions are capable of moving across the axonal membrane only at the myelin-free nodes (16). Thus a depolarization at one node spreads rapidly to the next node, and the action potential jumps from node to node. For this reason, the conduction velocity of myelinated nerves is much greater than that of unmyelinated nerves of the same diameter. For example, a 12-xcexcm-diameter myelinated vertebrate axon and a 600-xcexcm-diameter unmyelinated squid axon both conduct impulses at 12 m/s.
One of the more common neurologic diseases in human adults is multiple sclerosis. This condition is a chronic, frequently progressive, inflammatory CNS disease characterized pathologically by primary demyelination. The etiology and pathogenesis of multiple sclerosis are unknown. Researchers have hypothesized that multiple sclerosis is an autoimmune disease (14, 23, 47) or that a virus, bacteria or other agent, precipitates an inflammatory response in the CNS, which leads to either direct or indirect (xe2x80x9cbystanderxe2x80x9d) myelin destruction, potentially with an induced autoimmune component (31, 38). Thus, a rebuilding of the myelin sheath, or remyelination, can treat multiple sclerosis.
Spontaneous remyelination of axons within lesions by oligodendrocytes has been shown to occur to a small degree in SJL/J mice and multiple sclerosis patients (1). Several types of antibodies have been found to promote remyelination (1). Some of these antibodies are polyclonal, derived by immunization with spinal cord homogenate or myelin basic protein (71). One remyelination-promoting antibody is monoclonal (SCH 94.03) (1). The isotype of these antibodies is IgM, and they share the characteristic of binding to the surface of oligodendrocytes (1). Also, they are polyreactive, binding to a variety of cytoskeletal proteins or proteins with repeating structures (1).
Of clinical importance is the question whether morphologic regeneration of thin myelin sheaths contributes to functional recovery (1). Computer simulations indicate that new myelin formation even by inappropriately thin sheaths improves impulse conduction (1). Since the axon membrane of normally myelinated fibers is highly differentiated, it is necessary for sodium channels to be present at high density at the node of Ranvier to propagate saltatory conduction. Experimental evidence suggests that newly formed nodes do develop the required high sodium channel density as demonstrated by saxitoxin binding. Data suggest that remyelination even by inappropriately thin myelin improves conduction in a previously demyelinated axon. Therefore, any strategy to promote this morphologic phenomenon has the potential of producing functional recovery. Studies examining biopsy tissues from patients with severe acute exacerbations demonstrate that demyelination is a significant component of the acute multiple sclerosis lesion (57). Therefore, remissions are probably associated with significant CNS remyelination (1).
One commonly utilized experimental model of multiple sclerosis is induced by Theiler""s murine encephalomyelitis virus (TMEV) (15, 59). In the TMEV model, spinal cord demyelination is influenced by the immune response to virus infection and is therefore continuously sensitive to immunomodulation. Previous experiments in Strain Jackson Laboratories (SJL) mice infected with TMEV showed that 4 to 5% of the demyelinated area exhibited significant spontaneous remyelination (62). In protocols using antibody therapy and monoclonal antibody therapy, this number increased up to 30-35% (41, 58, 71). For instance, using the TMEV model, it was demonstrated that the passive transfer of CNS specific antiserum (63) and purified antibodies (55, 62, 71) directed against myelin components promoted CNS remyelination. This contrasts with the conventional view that the humoral immune response plays a pathogenic role in CNS demyelination (56). Researchers also generated a monoclonal antibody that reacted against a surface component of oligodendrocytes and promoted remyelination (40-42). It has also been shown that antibodies reactive with myelin basic protein (MBP) promoted CNS remyelination (58). In these experiments, infected SJL mice were treated with the whole anti-serum or affinity purified mouse antibodies directed against rabbit or rat myelin basic proteins. There was extensive evidence for new myelin synthesis as measured by quantitative morphometry. Electron microscopy revealed numerous oligodendrocytes and remyelinated CNS axons with a relative lack of inflammatory cells. Viral antigen persisted in these animals despite enhanced CNS remyelination. These findings indicated for the first time that antibodies reactive against a myelin autoantigen and in particular, MBP, have the potential for myelin repair.
U.S. Pat. No. 5,591,629 describes the promotion of CNS remyelination in the TMEV model through SCH 94.03 monoclonal antibodies directed against spinal cord homogenate (SCH) (1). SCH encompasses myelin antigens, such as MBP (64) and proteolipid protein (PLP) (12, 67). Although SCH contains MBP, this antibody does not react with MBP. The SCH 94.03 antibody is an IgM which recognizes cytoplasmic determinants on glial cells. It also recognizes surface determinants on glial cells, including oligodendrocytes. Experiments demonstrated that the antibody does not react with TMEV. In addition, the antibody was shown to promote the proliferation of glial cells in mixed rat brain culture in a dose-dependent manner. SCH 94.03 is a natural autoantibody.
A treatment that has been shown to be effective in reducing exacerbations of multiple sclerosis is the administration of glatiramer acetate (2-6, 31). Daily subcutaneous injections of glatiramer acetate (20 mg/injection) reduce relapse rates, appearance of new lesions by magnetic resonance imaging (MRI), and progression of disability (26). COPAXONE(copyright) is the brand name for glatiramer acetate (also known as Copolymer-1 (77), Copolymer 1, Cop-1 or Cop), an FDA-approved drug for the treatment of multiple sclerosis. Glatiramer acetate, the active ingredient of COPAXONE(copyright), consists of the acetate salts of synthetic polypeptides, containing four naturally occurring amino acids: L-glutamic acid, L-alanine, L-tyrosine, and L-lysine (77) with an average molar fraction of L-glutamic acid: 0.129-0.153; L-alanine: 0.392-0.462; L-tyrosine: 0.086-0.100; L-lysine: 0.300-0.376, respectively. The average molecular weight of glatiramer acetate is 4,700-11,000 daltons (77). Chemically, glatiramer acetate is designated L-glutamic acid polymer with L-alanine, L-lysine and L-tyrosine, acetate (salt) (77). Its structural formula is:
(Glu, Ala, Lys, Tyr)x.CH3COOH(C5H9NO4.C3H7NO2.C6H14N2O2.C9H11NO3)xcex."khgr"C2H4O2CAS-147245-92-9
(77). Glatiramer acetate is also written as: poly[L-Glu13-15, L-Ala39-46, L-Tyr8.6-10, L-Lys30-37]nCH3COOH.
Unlike myelin basic protein (MBP), with which it shares some structural characteristics, glatiramer acetate inhibits rather than induces experimental autoimmune encephalomyelitis (EAE), an animal model of MS (37, 65-66). Glatiramer acetate-reactive, type 2 helper T lymphocytes confer resistance to EAE.
In spite of the experimental evidence that glatiramer acetate down-regulates certain immune functions, clinical use of glatiramer acetate indicates that other immune functions are stimulated by the peptide treatment. In rodents, monoclonal antibodies to glatiramer acetate have been generated, some of which cross-react with MBP (68), but other cross-reactivities are unknown. The humoral response to glatiramer acetate may haye diverse roles in multiple sclerosis. Some autoreactive antibodies to myelin antigens might contribute to pathogenesis (22, 35). Other antibodies, such as those that develop in a subset of interferon-treated patients, may neutralize therapeutic efficacy. A third possibility is that some antibodies may in fact be protective. All individuals have antibodies to a wide range of endogenous antigens, including MBP, suggesting that natural autoantibodies represent a conserved adaptation to nervous system disease and trauma. In support of a protective role for autoreactive antibodies, mouse or human antibodies reactive to the central nervous system (CNS) have been found to promote myelin repair in viral experimental model of multiple sclerosis (41, 48, 53, 58). Antibodies have been found to stimulate remyelination in SJL mice that were chronically infected with EAE (41, 58).
Antisera against glatiramer acetate have been employed to investigate the mechanism by which L-glatiramer acetate is effective against Experimental Allergic Encephalomyelitis (EAE) (74-75). For this purpose, Webb et al. measured the cross-reactivity of L-glatiramer acetate anti-sera with D-glatiramer acetate and Copolymer 4 (L-glatiramer acetate modified by the replacement of tyrosine with tryptophan) (75). Webb et al. carried out a similar experiment to determine the reactivity of L-glatiramer acetate anti-sera with L-glatiramer acetate, and the cross-reactivity of L-glatiramer acetate anti-sera with AGT (alanine, glutamic acid and tyrosine), BE (Basic Encephalitogen), AAspLT (alanine, aspartic acid, lysine and tyrosine) and AGL (alanine, glutamic acid and lysine) (74).
Monoclonal antibodies against glatiramer acetate and against MBP have also been utilized to probe the mechanism of glatiramer acetate in treatment of EAE (68). The cross-reactivity of monoclonal antibodies against glatiramer acetate with MBP was analyzed by Teitelbaum et al (68). They also determined the cross-reactivity of monoclonal antibodies against MBP with glatiramer acetate (68). Another focus of their experiments was the cross-reactivity of glatiramer acetate anti-sera with MBP and of MBP-antisera with glatiramer acetate (68). The cross-reactivity of anti-MBP anti-sera with glatiramer acetate was additionally investigated by Lisak et al (37).
The subject invention concerns a humanized antibody directed against an epitope on glatiramer acetate, also known as Copolymer 1, Copolymer-1, Cop-1 or Cop.
The subject invention further encompasses a Fab fragment that binds to an epitope on glatiramer acetate.
In addition, the subject invention relates to a pharmaceutical composition comprising an antibody directed against an epitope on glatiramer acetate in an amount effective to treat a central nervous system disease and a pharmaceutically acceptable carrier.
The subject invention also provides a method of stimulating remyelination of central nervous system axons comprising contacting the axons with an antibody directed against an epitope on glatiramer acetate in an amount effective to stimulate remyelination of central nervous system axons.
The subject invention additionally includes a method of treating a subject suffering from a disease associated with demyelination of central nervous system axons comprising administering to the subject an effective amount of an antibody directed against an epitope on glatiramer acetate in an amount effective to treat the disease associated with demyelination of central nervous system axons.
The subject invention further relates to a method of stimulating remyelination of central nervous system axons comprising contacting the axons with glatiramer acetate in an amount effective to stimulate remyelination of central nervous system axons.
The subject invention also concerns a method of treating a subject suffering from a disease associated with demyelination of central nervous system axons comprising administering to the subject glatiramer acetate in an amount effective to treat the disease associated with demyelination of central nervous system axons, wherein the disease associated with demyelination of central nervous system axons is selected from the group consisting of: acute disseminated encephalomyelitis, transverse myelitis, demyelinating genetic diseases, spinal cord injury, virus-induced demyelination, Progressive Multifocal Leucoencephalopathy, Human Lymphotrophic T-cell Virus I (HTLVI)-associated myelopathy, and nutritional metabolic disorders.
Finally, the subject invention encompasses a method of stimulating proliferation of lymphocytes comprising contacting the lymphocytes with an antibody directed against an epitope on glatiramer acetate in an amount effective to stimulate lymphocyte proliferation.